Retrospective insights of the COVID-19 epidemic in the major latin American city, São Paulo, Southeastern Brazil

São Paulo is the financial center of Brazil, with a population of over 12 million, that receives travelers from all over the world for business and tourism. It was the first city in Brazil to report a case of COVID-19 that rapidly spread across the city despite the implementation of the restriction measures. Despite many reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the city of São Paulo. Thus, in this study, we provide a retrospective overview of the COVID-19 epidemic in São Paulo City, Southeastern, Brazil, by generating a total of 9995 near-complete genome sequences from all the city’s different macro-regions (North, West, Central, East, South, and Southeast). Our analysis revealed that multiple independent introduction events of different variants (mainly Gamma, Delta, and Omicron) occurred throughout time. Additionally, our estimates of viral movement within the different macro-regions further suggested that the East and the Southeast regions were the largest contributors to the Gamma and Delta viral exchanges to other regions. Meanwhile, the North region had a higher contribution to the dispersion of the Omicron variant. Together, our results reinforce the importance of increasing SARS-CoV-2 genomic monitoring within the city and the country to track the real-time evolution of the virus and to detect earlier any eventual emergency of new variants of concern that could undermine the fight against COVID-19 in Brazil and worldwide.

A traveling SARS-CoV-2 laboratory as part of a pandemic response among vulnerable Brazilian populations

Background Brazil has been dramatically hit by the SARS-CoV-2 pandemic and is a world leader in COVID-19 morbidity and mortality. Additionally, the largest country of Latin America has been a continuous source of SARS-CoV-2 variants and shows extraordinary variability of the pandemic strains probably related to the country´s outstanding position as a Latin American economical and transportation hub. Not all regions of the country show sufficient infrastructure for SARS-CoV-2 diagnosis and genotyping which can negatively impact the pandemic response. Methods Due to this reason and to disburden the diagnostic system of the inner São Paulo State, the Butantan Institute established the Mobile Laboratory (in Portuguese: LabMovel) for SARS-CoV-2 testing which started a trip of the most important “hotspots” of the most populous Brazilian region. The LabMovel initiated in two important cities of the State: Aparecida do Norte (an important religious center) and the Baixada Santista region which incorporates the port of Santos, the busiest in Latin America. The LabMovel was fully equipped with an automatized system for SARS-CoV-2 diagnosis and sequencing/genotyping. It also integrated the laboratory systems for patient records and results divulgation including in the Federal Brazilian Healthcare System. Results Currently,16,678 samples were tested, among them 1,217 from Aparecida and 4,564 from Baixada Santista. We tracked the delta introductio in the tested regions with its high diversification. The established mobile SARS-CoV-2 laboratory had a major impact on the Public Health System of the included cities including timely delivery of the results to the healthcare agents and the Federal Healthcare system, evaluation of the vaccination status of the positive individuals in the background of exponential vaccination process in Brazil and scientific and technological divulgation of the fieldwork to the most vulnerable populations. Conclusions The SARS-CoV-2 pandemic has demonstrated worldwide the importance of science to fight against this viral agent and the LabMovel shows that it is possible to integrate researchers, clinicians, healthcare workers and patients to take rapid actions that can in fact mitigate this and other epidemiological situations.

Genomic epidemiology of the SARS-CoV-2 epidemic in Brazil

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.

Genomic epidemiology of the SARS-CoV-2 epidemic in Brazil

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.

The divergent pattern of SARS-CoV-2 variant predominance and transmission dynamics in the Brazilian island of Ilhabela

Our effort in SARS-CoV-2 genomic surveillance in Brazil has detected the Alpha Variant of Concern with a predominance higher than 75% in the population of Ilhabela island (São Paulo State) at a time when the Gamma VOC was already predominating the mainland raised concerns for closer surveillance on this island. Therefore, we intensified the surveillance for 24 weeks by generating data from 34% of local positive cases. Our data show that the patterns of VOC predominance dynamics and infection rates were in general distinct from the mainland. We report here the first known case of Alpha predominance in a Brazilian population, a delay greater than 3 months for the Gamma to dominate the previous variants compared to the mainland, and a faster dispersion rate of Gamma and Delta VOCs compared to the mainland. Phylogenetic analysis revealed the SARS-CoV-2 transmission dynamics in Ilhabela were characterized by multiple independent introduction events of Gamma and Delta, with a few events of Alpha introduction, two of them followed by community transmission. This study evidenced the peculiar behavior of SARS-CoV-2 variants in an isolated population and brought to light the importance of specific programs for SARS-CoV-2 genomic surveillance in isolated populations.

SARS-COV-2 genomic monitoring in the state of São Paulo unveils two emerging AY.43 sublineages

Delta VOC is highly diverse with more than 120 sublineages already described as of November 30, 2021. In this study, through active monitoring of circulating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants in the state of São Paulo, southeast Brazil, we identified two emerging sublineages from the ancestral AY.43 strain which were classified as AY.43.1 and AY.43.2. These sublineages were defined by the following characteristic nonsynonymous mutations ORF1ab:A4133V and ORF3a:T14I for the AY.43.1 and ORF1ab:G1155C for the AY.43.2 and our analysis reveals that they might have a likely-Brazilian origin. Much is still unknown regarding their dissemination in the state of São Paulo and Brazil as well as their potential impact on the ongoing vaccination process. However, the results obtained in this study reinforce the importance of genomic surveillance activity for timely identification of emerging SARS-CoV-2 variants which can impact the ongoing SARS-CoV-2 vaccination and public health policies.

Genomic monitoring of the SARS-CoV-2 B1.1.7 (WHO VOC Alpha) in the Sao Paulo state, Brazil

The SARS-CoV-2 alpha VOC (also known as lineage B.1.1.7) initially described in the autumn, 2020 in UK, rapidly became the dominant lineage across much of Europe. Despite multiple studies reporting molecular evidence suggestive of its circulation in Brazil, much is still unknown about its genomic diversity in the state of São Paulo, the main Brazilian economic and transportation hub. To get more insight regarding its transmission dynamics into the State we performed phylogenetic analysis on all alpha VOC strains obtained between February and August 2021 from the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants. The performed phylogenetic analysis showed that most of the alpha VOC genomes were interspersed with viral strains sampled from different Brazilian states and other countries suggesting that multiple independent Alpha VOC introductions from Brazil and overseas have occurred in the São Paulo State over time. Nevertheless, large monophyletic clusters were also observed especially from the Central-West part of the São Paulo State (the city of Bauru) and the metropolitan region of the São Paulo city. Our results highlight the Alpha VOC molecular epidemiology in the São Paulo state and reinforce the need for continued genomic surveillance strategies for the real-time monitoring of potential emerging SARS-CoV-2 variants during the ever-growing vaccination process.

Nucleocapsid (N) gene mutations of SARS-CoV-2 can affect real-time RT-PCR diagnostic and impact false-negative results

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

SARS-CoV-2 genomic monitoring in the São Paulo State unveils new sublineages of the AY.43 strain

Delta VOC is highly diverse and more than 120 sublineages have been identified in Pango lineages with the continuous description of emerging ones. Brazil is now one of the most vaccinated countries against SARS-CoV-2 in the world which can enhance the emergence of viral mutations related to improved viral fitness. In this study, we identified two novel sublineages of the AY.43 lineage which were classified as AY.43.1 and AY.43.2 as observed on the specific clustering on the obtained phylogenetic tree. The novel sublineages were defined by the following characteristic nonsynonymous mutations ORF1ab:A4133V and ORF3a:T14I for AY.43.1 and ORF1ab:G1155C for AY.43.2. The majority of the analyzed sequences of both lineages were Brazilian, which shows that probably these two emerging sublineages have Brazilian origin. It is still unknown how these two sublineages are disseminated in São Paulo State and Brazil and their potential impact on the ongoing vaccination process. However, the performed study reinforces the importance of the SARS-CoV-2 genome monitoring for timely identification of emerging SARS-CoV-2 variants which can impact the ongoing SARS-CoV-2 vaccination and public health policies.

Genomic monitoring unveil the early detection of the SARS-CoV-2 B.1.351 (beta) variant (20H/501Y.V2) in Brazil

Sao Paulo State, currently experiences a second COVID-19 wave overwhelming the healthcare system. Due to the paucity of SARS-CoV-2 complete genome sequencing, we established a Network for Pandemic Alert of Emerging SARS-CoV-2 Variants to rapidly understand and monitor the spread of SARS-CoV-2 variants into the state. Through analysis of 210 SARS-CoV-2 complete genomes obtained from the largest regional health departments we identified cocirculation of multiple SARS-CoV-2 lineages such as B.1.1 (0.5%), B.1.1.28 (23.2%), B.1.1.7 (alpha variant, 6.2%), B.1.566 (1.4%), B.1.544 (0.5%), C.37 (0.5%) P.1 (gamma variant, 66.2%), and P.2 (zeta variant, 1.0%). Our analysis allowed also the detection, for the first time in Brazil, the South African B.1.351 (beta) variant of concern, B.1.351 (501Y.V2) 0.5%, characterized by the following mutations: ORF1ab: T265I, R724K, S1612L, K1655N, K3353R, SGF 3675_F3677del, P4715L, E5585D; spike: D80A, D215G, L242_L244del, A262D, K417N, E484K, N501Y, D614G, A701V, C1247F; ORF3a: Q57H, S171L, E: P71L; ORF7b: Y10F, N: T205I; ORF14: L52F. The most recent common ancestor of the identified strain was inferred to be mid-October to late December 2020. Our analysis demonstrated the P.1 lineage predominance and allowed the early detection of the South African strain for the first time in Brazil. We highlight the importance of SARS-CoV-2 active monitoring to ensure the rapid detection of potential variants for pandemic control and vaccination strategies.

Colostrum from primiparous Holstein cows shows higher antioxidant activity than colostrum of multiparous ones.

Antioxidant components of colostrum prevent oxidative cell damage caused by free radicals that could harm the calf's development. The relationship of antioxidant potential of colostrum with parity is not well defined and could enlighten the importance of these components for the neonate and for the protection of the intestinal epithelium. The purpose of this work was to determine the antioxidant potential of colostrum from primiparous and multiparous Holstein cows in a commercial dairy farm. Samples from the first milk secretion from primiparous (first lactation, n = 8) and multiparous (second and third lactations, n = 8) Holstein cows were collected after birth of calves for determination of immune and antioxidant factors. The cows sampled in this study were vaccinated during pregnancy in order to improve colostrum quality. Colostrum from primiparous cows showed higher values of ceruloplasmin activity, oxygen radical absorbance capacity (ORAC) and transferrin saturation index (TSI) than colostrum from multiparous cows (P < 0.05). The total iron binding capacity (TIBC) and transferrin concentration in the colostrum of primiparous cows showed a non-significant numerical decrease (P = 0.06) in relation to the value in the colostrum of multiparous cows. Concentration of proteins, immunoglobulin G, and activity of lactoperoxidase, lysozyme, glutathione peroxidase and catalase, in turn, did not differ (P > 0.05). Metabolic differences between primiparous and multiparous cows may have affected the antioxidative status of colostrum, since ORAC values were twice higher in first lactation cows. Lower values of transferrin and TIBC and higher TSI in colostrum from primiparous cows suggests a relationship between lower iron stock and higher antioxidant activity. Thus, this work indicates an important role of the antioxidant potential of colostrum for neonates from first-lactation cows. Additionally, the iron stock may be directly related to the higher antioxidant potential of the colostrum from primiparous cows, and further investigations are required.

Long-term chromium picolinate supplementation improves colostrum profile of Santa Ines ewe.

Chromium (Cr) is a micromineral that is involved in the metabolism of carbohydrates, lipids, ammonia, and nucleic acids; thus, its supplementation can influence the nutritional status of ruminants, and consequently, colostrum profile, since this secretion depends on products secreted by the mammary gland and elements of the maternal bloodstream. The present study investigated the influence of supplementation with Cr bound to organic molecule on the nutritional, immune, and antioxidant quality of ewe colostrum. Thirty-two multiparous Santa Ines ewes (55.3 ± 8.00 kg body weight) were randomly assigned into four groups: T1 (0.0 mg of chromium picolinate (CrPic) supplementation per ewe, n = 8), T2 (0.15 mg of CrPic per ewe, n = 9), T3 (0.30 mg of CrPic per ewe, n = 7), and T4 (0.45 mg of CrPic per ewe, n = 8). Supplementation was supplied during the breeding season, pregnancy, and lactation. Shortly after calving, the first milking colostrum was collected to determine its chemical composition, activity of lysozyme, lactoperoxidase, ceruloplasmin, catalase, glutathione peroxidase, and oxygen radical absorbance capacity. The results show that lactoperoxidase activity decreased with CrPic supplementation (P < 0.01), revealing that this micromineral reduces an important component of defense mechanism in the body. Therefore, the results of this work show that supplementation with chromium picolinate influences colostrum quality.

Cottonseed feeding changes oxidative stress markers in ewes during the peripartum period and increases the quality of colostrum.

Immune indicators and oxidative stress markers were analyzed in blood and lacteal secretions of twenty Santa Ines ewes distributed in two feeding regimes: cottonseed concentrate (CS; n=10) and soybean concentrate (SB; n=10). Blood catalase activity and oxygen radical absorbance capacity were higher in SB than CS (P<0.05), suggesting that cottonseed increased oxidative stress in ewes. Cottonseed consumption increased IgG concentration, lysozyme activity and total protein concentration in the colostrum and decreased iron stock, suggesting a positive effect of this nutrient on the immune quality, antibacterial activity and antioxidant potential of colostrum.

Effect of maternal cottonseed feed on the immune and antioxidant status of Santa Ines lambs.

Cottonseed has been used as a nutritional alternative in animal production. However, consequences of this nutrient in the progeny is not well characterized. Thus, this work evaluated the effect on the immune and antioxidant status of the progeny of feeding Santa Ines ewe with or without cottonseed. Twenty-four Santa Ines ewes were distributed in two feeding regimes: cottonseed (CS) concentrate (n = 12) and soybean (SB) concentrate (n = 12). After birth, lambs remained with their mothers and blood samples were collected at 1st, 3rd, 7th, 15th, 30th and 60th day of life of 24 lambs born from mothers fed with (CS, n = 12) or without (SB, n = 12) cottonseed. Serum total protein, albumin, alpha beta globulin, gamma globulin, immunoglobulin G and M, activity of glutathione peroxidase (GPx), catalase (CAT), oxygen radical absorbance capacity (ORAC) and variables related to iron metabolism were affected only by sampling times (P < 0.05). The concentration of serum total protein, alpha beta globulin, gamma globulin and immunoglobulin G and M, GPx activity and ORAC values decreased as lamb age increased. Serum albumin concentration and CAT activity, in turn, increased as lamb age increased. In this work, maternal feeding with cottonseed did not affect the serum protein profile and antioxidant status of progeny during the lactation period, indicating no transfer of gossypol effects by milk secretions. Thus, the alternative in ruminants feeding with cottonseed can be used without maternal-descendant effects to immunity and oxidative stress in lambs.

Morphological-metric, ultrastructural and immunohistochemical effects of gossypol on cultured granulosa cells and oocytes of ewes using MOEPF.

Manipulation of oocytes enclosed in preantral follicles (MOEPF) allows for analyzing follicular development and use of this biotechnology in the pre-analysis of the beneficial or toxic effects of bio-products on granulosa cells and oocytes at different developmental stages. In this study, there was evaluation of the effects of gossypol by culturing granulosa cells and oocytes in ewe ovarian tissues. Ovarian tissues were cultured with gossypol at 37 °C, in humidified air and 5% CO2. Variables that were evaluated were morphology, morphometry, ultrastructure and abundance of estradiol receptor α (α-ER). There were no differences in developmental characteristics when there was treatment with any of the gossypol doses that were evaluated. Immunostaining indicated that when the gossypol dose increases, the abundance of α-ER also increases in the cytoplasm, nucleus, and granulosa cells. Findings with the ultrastructural analysis indicated that for granulosa cells there was fewer cells and greater disorganization and a lack of structural integrity of follicular cell layers as a result of all gossypol treatments. The culture of oocytes in preantral ovarian follicles in presence of gossypol did not affect the morphological-metric structure at the doses evaluated. The findings with evaluation of ultrastructural and immunohistochemical structures indicated granulosa cells and α-ER were affected by the treatments with gossypol indicating there were effects of this compound on ovarian function in sheep. This study indicated there is a toxic action of gossypol when using the biotechnology, MOEPF. Thus, gossypol negatively affects granulosa cell development and structural integrity of preantral follicles in sheep.

Histochemical distribution of intestinal enzymes of juvenile pacu (Piaractus mesopotamicus) fed lyophilized bovine colostrum.

Enzyme activity was evaluated in the intestine of juvenile pacu, Piaractus mesopotamicus, fed diets containing 0, 10 or 20 % of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The enzymes intestinal acid and alkaline phosphatase (ACP and ALP, respectively), nonspecific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and rectum). Moderate activity of the DAP IV was detected in the three last intestinal segments, but no differences among the treatments were detected. Enzymes LAP, NSE and LIP were weakly stained in all intestinal segments and the inclusion of 10 or 20 % of LBC in the diet commanded a moderate reaction to NSE in the S3 segment at day 60. ACP activity was detected only in the brush border of the S1 segment of fish fed 0 % LBC for either 30 or 60 days. The activity of ALP was very strong in the first intestinal segment, but a weak reaction was seen in the last segments. The inclusion of 20 % of LBC changed the pattern of staining to the ALP, eliciting moderate staining in S2 at day 30 and S1 at day 60. The consumption of diets containing LBC by juvenile pacu did not have significant implications in intestinal enzymatic activity, which still was not fully stimulated.